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1.
J Am Chem Soc ; 146(28): 19088-19100, 2024 Jul 17.
Artigo em Inglês | MEDLINE | ID: mdl-38946086

RESUMO

Antibody-drug conjugates (ADCs) for the treatment of cancer aim to achieve selective delivery of a cytotoxic payload to tumor cells while sparing normal tissue. In vivo, multiple tumor-dependent and -independent processes act on ADCs and their released payloads to impact tumor-versus-normal delivery, often resulting in a poor therapeutic window. An ADC with a labeled payload would make synchronous correlations between distribution and tissue-specific pharmacological effects possible, empowering preclinical and clinical efforts to improve tumor-selective delivery; however, few methods to label small molecules without destroying their pharmacological activity exist. Herein, we present a bioorthogonal switch approach that allows a radiolabel attached to an ADC payload to be removed tracelessly at will. We exemplify this approach with a potent DNA-damaging agent, the pyrrolobenzodiazepine (PBD) dimer, delivered as an antibody conjugate targeted to lung tumor cells. The radiometal chelating group, DOTA, was attached via a novel trans-cyclooctene (TCO)-caged self-immolative para-aminobenzyl (PAB) linker to the PBD, stably attenuating payload activity and allowing tracking of biodistribution in tumor-bearing mice via SPECT-CT imaging (live) or gamma counting (post-mortem). Following TCO-PAB-DOTA reaction with tetrazines optimized for extra- and intracellular reactivity, the label was removed to reveal the unmodified PBD dimer capable of inducing potent tumor cell killing in vitro and in mouse xenografts. The switchable antibody radio-drug conjugate (ArDC) we describe integrates, but decouples, the two functions of a theranostic given that it can serve as a diagnostic for payload delivery in the labeled state, but can be switched on demand to a therapeutic agent (an ADC).


Assuntos
Imunoconjugados , Tomografia Computadorizada de Emissão de Fóton Único , Imunoconjugados/química , Humanos , Animais , Camundongos , Benzodiazepinas/química , Linhagem Celular Tumoral , Antineoplásicos/química , Antineoplásicos/farmacologia , Pirróis/química
3.
Eur J Nucl Med Mol Imaging ; 50(2): 287-301, 2023 01.
Artigo em Inglês | MEDLINE | ID: mdl-36271158

RESUMO

BACKGROUND: ZED8 is a novel monovalent antibody labeled with zirconium-89 for the molecular imaging of CD8. This work describes nonclinical studies performed in part to provide rationale for and to inform expectations in the early clinical development of ZED8, such as in the studies outlined in clinical trial registry NCT04029181 [1]. METHODS: Surface plasmon resonance, X-ray crystallography, and flow cytometry were used to characterize the ZED8-CD8 binding interaction, its specificity, and its impact on T cell function. Immuno-PET with ZED8 was assessed in huCD8+ tumor-bearing mice and in non-human primates. Plasma antibody levels were measured by ELISA to determine pharmacokinetic parameters, and OLINDA 1.0 was used to estimate radiation dosimetry from image-derived biodistribution data. RESULTS: ZED8 selectively binds to human CD8α at a binding site approximately 9 Å from that of MHCI making mutual interference unlikely. The equilibrium dissociation constant (KD) is 5 nM. ZED8 binds to cynomolgus CD8 with reduced affinity (66 nM) but it has no measurable affinity for rat or mouse CD8. In a series of lymphoma xenografts, ZED8 imaging was able to identify different CD8 levels concordant with flow cytometry. In cynomolgus monkeys with tool compound 89Zr-aCD8v17, lymph nodes were conspicuous by imaging 24 h post-injection, and the pharmacokinetics suggested a flat-fixed first-in-human dose of 4 mg per subject. The whole-body effective dose for an adult human was estimated to be 0.48 mSv/MBq, comparable to existing 89Zr immuno-PET reagents. CONCLUSION: 89Zr immuno-PET with ZED8 appears to be a promising biomarker of tissue CD8 levels suitable for clinical evaluation in cancer patients eligible for immunotherapy.


Assuntos
Neoplasias , Tomografia por Emissão de Pósitrons , Adulto , Humanos , Camundongos , Ratos , Animais , Tomografia por Emissão de Pósitrons/métodos , Indicadores e Reagentes/uso terapêutico , Distribuição Tecidual , Neoplasias/terapia , Neoplasias/tratamento farmacológico , Imunoterapia/métodos , Zircônio/química , Linfócitos T CD8-Positivos/metabolismo , Linhagem Celular Tumoral
4.
Mol Cancer Ther ; 20(10): 2008-2015, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34315765

RESUMO

Advances in antibody engineering have enabled the construction of novel molecular formats in diverse shapes and sizes, providing new opportunities for cancer immunotherapeutic drug discovery while also revealing limitations in knowledge of structure-activity relationships. The current understanding of renal filtration originates largely from data reported for dextrans, IgG, albumin, and selected globular proteins. For a one-armed IgG-based T-cell imaging agent, we observed higher renal signal than typically observed for bivalent IgGs, prompting us to explore the factors governing renal filtration of biologics. We constructed a small representative library of IgG-like formats with varied shapes and hinge flexibilities falling broadly into two categories: branched molecules including bivalent IgG and (scFv)2Fc, and nonbranched molecules including one-armed IgG, one-armed IgG with stacked Fab, and one-armed IgG with a rigid IgA2 hinge. Transmission electron microscopy revealed Y-shaped structures for the branched molecules and pseudo-linear structures for the nonbranched molecules. Single-photon emission CT imaging, autoradiography, and tissue harvest studies demonstrated higher renal uptake and catabolism for nonbranched molecules relative to branched molecules. Among the nonbranched molecules, the one-armed IgG with rigid IgA2 hinge molecule demonstrated higher kidney uptake and decreased systemic exposure relative to molecules with a more flexible hinge. Our results show that differences in shape and hinge flexibility drive the increased glomerular filtration of one-armed relative to bivalent antibodies and highlight the practical advantages of using imaging to assess renal filtration properties. These findings are particularly relevant for T-cell-dependent bispecific molecules, many of which have nonstandard antibody structures.


Assuntos
Anticorpos Biespecíficos/imunologia , Anticorpos Monoclonais/imunologia , Barreira de Filtração Glomerular/metabolismo , Imunoglobulina G/imunologia , Tomografia Computadorizada com Tomografia Computadorizada de Emissão de Fóton Único/métodos , Proteínas do Envelope Viral/imunologia , Animais , Anticorpos Biespecíficos/administração & dosagem , Anticorpos Monoclonais/administração & dosagem , Feminino , Barreira de Filtração Glomerular/efeitos dos fármacos , Humanos , Imunoglobulina G/classificação , Camundongos SCID
5.
Mol Cancer Ther ; 20(10): 1956-1965, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34253591

RESUMO

T-cell-dependent bispecific antibodies (TDB) have been a major advancement in the treatment of cancer, allowing for improved targeting and efficacy for large molecule therapeutics. TDBs are comprised of one arm targeting a surface antigen on a cancer cell and another targeting an engaging surface antigen on a cytotoxic T cell. To impart this function, the antibody must be in a bispecific format as opposed to the more conventional bivalent format. Through in vitro and in vivo studies, we sought to determine the impact of changing antibody valency on solid tumor distribution and catabolism. A bivalent anti-HER2 antibody exhibited higher catabolism than its full-length monovalent binding counterpart in vivo by both invasive tissue harvesting and noninvasive single photon emission computed tomography/X-ray computed tomography imaging despite similar systemic exposures for the two molecules. To determine what molecular factors drove in vivo distribution and uptake, we developed a mechanistic model for binding and catabolism of monovalent and bivalent HER2 antibodies in KPL4 cells. This model suggests that observed differences in cellular uptake of monovalent and bivalent antibodies are caused by the change in apparent affinity conferred by avidity as well as differences in internalization and degradation rates of receptor bound antibodies. To our knowledge, this is the first study to directly compare the targeting abilities of monovalent and bivalent full-length antibodies. These findings may inform diverse antibody therapeutic modalities, including T-cell-redirecting therapies and drug delivery strategies relying upon receptor internalization.


Assuntos
Anticorpos Biespecíficos/farmacologia , Anticorpos Biespecíficos/farmacocinética , Afinidade de Anticorpos , Neoplasias da Mama/tratamento farmacológico , Receptor ErbB-2/antagonistas & inibidores , Linfócitos T Citotóxicos/imunologia , Animais , Anticorpos Biespecíficos/imunologia , Apoptose , Neoplasias da Mama/imunologia , Neoplasias da Mama/patologia , Proliferação de Células , Feminino , Humanos , Camundongos , Camundongos SCID , Receptor ErbB-2/imunologia , Distribuição Tecidual , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto
6.
Front Pharmacol ; 12: 601569, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34025395

RESUMO

Protein therapeutics have witnessed tremendous use and application in recent years in treatment of various diseases. Predicting efficacy and safety during drug discovery and translational development is a key factor for successful clinical development of these therapies. In general, drug related toxicities are predominantly driven by pharmacokinetic (PK) exposure at off-target sites. This work explores the ocular PK of intravenously administered protein therapeutics to understand impact of antibody format on off-site exposure. Species matched non-binding rabbit antibody proteins (rabFab and rabIgG) were intravenously administered to male New Zealand White rabbits at a single 1 mg bolus dose and exposure was measured up to 3 weeks. As anticipated based on absence of FcRn recycling, rabFab has relatively fast systemic PK (CL-943 mL/day and t1/2-1.93 days) compared to rabIgG (CL-18.5 mL/day and t1/2-8.93 days). Similarly, rabFab has lower absolute ocular exposure in ocular compartments (e.g., vitreous and aqueous humor) compared to rabIgG, despite higher relative exposures (measured as percent tissue partition in ocular tissues relative to serum, based on Cmax and AUC). In general, percent tissue partition based on AUC (in aqueous and vitreous humor) relative to serum exposure were 10.4 and 8.62 for rabFab respectively and 1.11 and 0.64 for rabIgG respectively. This work emphasizes size and format based ocular exposure of intravenously administered protein therapeutics. Findings from this work enable prediction of format based ocular exposure for systemically administered antibody based therapeutics and aid in selection of molecule format for clinical candidate to minimize ocular exposure.

7.
Mol Cancer Ther ; 20(4): 716-725, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33536191

RESUMO

Ovarian cancer is a diverse class of tumors with very few effective treatment options and suboptimal response rates in early clinical studies using immunotherapies. Here we describe LY6/PLAUR domain containing 1 (LYPD1) as a novel target for therapeutic antibodies for the treatment of ovarian cancer. LYPD1 is broadly expressed in both primary and metastatic ovarian cancer with ∼70% prevalence in the serous cancer subset. Bispecific antibodies targeting CD3 on T cells and a tumor antigen on cancer cells have demonstrated significant clinical activity in hematologic cancers. We have developed an anti-LYPD1/CD3 T-cell-dependent bispecific antibody (TDB) to redirect T-cell responses to LYPD1 expressing ovarian cancer. Here we characterize the nonclinical pharmacology of anti-LYPD1/CD3 TDB and show induction of a robust polyclonal T-cell activation and target dependent killing of LYPD1 expressing ovarian cancer cells resulting in efficient in vivo antitumor responses in PBMC reconstituted immune-deficient mice and human CD3 transgenic mouse models. Anti-LYPD1/CD3 TDB is generally well tolerated at high-dose levels in mice, a pharmacologically relevant species, and showed no evidence of toxicity or damage to LYPD1 expressing tissues.


Assuntos
Anticorpos Biespecíficos/uso terapêutico , Complexo CD3/imunologia , Neoplasias Ovarianas/tratamento farmacológico , Sequência de Aminoácidos , Animais , Anticorpos Biespecíficos/farmacologia , Linhagem Celular Tumoral , Feminino , Humanos , Camundongos , Camundongos Transgênicos , Neoplasias Ovarianas/patologia
8.
MAbs ; 13(1): 1862452, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33382956

RESUMO

Early success with brentuximab vedotin in treating classical Hodgkin lymphoma spurred an influx of at least 20 monomethyl auristatin E (MMAE) antibody-drug conjugates (ADCs) into clinical trials. While three MMAE-ADCs have been approved, most of these conjugates are no longer being investigated in clinical trials. Some auristatin conjugates show limited or no efficacy at tolerated doses, but even for drugs driving initial remissions, tumor regrowth and metastasis often rapidly occur. Here we describe the development of second-generation therapeutic ADCs targeting Lymphocyte antigen 6E (Ly6E) where the tubulin polymerization inhibitor MMAE (Compound 1) is replaced with DNA-damaging agents intended to drive increased durability of response. Comparison of a seco-cyclopropyl benzoindol-4-one (CBI)-dimer (compound 2) to MMAE showed increased potency, activity across more cell lines, and resistance to efflux by P-glycoprotein, a drug transporter commonly upregulated in tumors. Both anti-Ly6E-CBI and -MMAE conjugates drove single-dose efficacy in xenograft and patient-derived xenograft models, but seco-CBI-dimer conjugates showed reduced tumor outgrowth following multiple weeks of treatment, suggesting that they are less susceptible to developing resistance. In parallel, we explored approaches to optimize the targeting antibody. In contrast to immunization with recombinant Ly6E or Ly6E DNA, immunization with virus-like particles generated a high-affinity anti-Ly6E antibody. Conjugates to this antibody improve efficacy versus a previous clinical candidate both in vitro and in vivo with multiple cytotoxics. Conjugation of compound 2 to the second-generation antibody results in a substantially improved ADC with promising preclinical efficacy.


Assuntos
Anticorpos Monoclonais/imunologia , Antígenos de Superfície/imunologia , Antineoplásicos/imunologia , Imunoconjugados/imunologia , Oligopeptídeos/imunologia , Ensaios Antitumorais Modelo de Xenoenxerto/métodos , Animais , Anticorpos Monoclonais/farmacocinética , Anticorpos Monoclonais/farmacologia , Antineoplásicos/farmacocinética , Antineoplásicos/farmacologia , Antineoplásicos Imunológicos/farmacocinética , Antineoplásicos Imunológicos/farmacologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/imunologia , Feminino , Proteínas Ligadas por GPI/imunologia , Células HEK293 , Humanos , Imunoconjugados/farmacocinética , Imunoconjugados/farmacologia , Camundongos SCID , Ratos Sprague-Dawley , Carga Tumoral/efeitos dos fármacos , Carga Tumoral/imunologia
9.
Mol Cancer Ther ; 19(4): 1052-1058, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32024685

RESUMO

Full-length antibodies lack ideal pharmacokinetic properties for rapid targeted imaging, prompting the pursuit of smaller peptides and fragments. Nevertheless, studying the disposition properties of antibody-based imaging agents can provide critical insight into the pharmacology of their therapeutic counterparts, particularly for those coupled with potent payloads. Here, we evaluate modulation of binding to the neonatal Fc receptor (FcRn) as a protein engineering-based pharmacologic strategy to minimize the overall blood pool background with directly labeled antibodies and undesirable systemic click reaction of radiolabeled tetrazine with circulating pretargeted trans-cyclooctene (TCO)-modified antibodies. Noninvasive SPECT imaging of mice bearing HER2-expressing xenografts was performed both directly (111In-labeled antibody) and indirectly (pretargeted TCO-modified antibody followed by 111In-labeled tetrazine). Pharmacokinetic modulation of antibodies was achieved by two distinct methods: Fc engineering to reduce binding affinity to FcRn, and delayed administration of an antibody that competes with binding to FcRn. Tumor imaging with directly labeled antibodies was feasible in the absence of FcRn binding, rapidly attaining high tumor-to-blood ratios, but accompanied by moderate liver and spleen uptake. Pretargeted imaging of tumors with non-FcRn-binding antibody was also feasible, but systemic click reaction still occurred, albeit at lower levels than with parental antibody. Our findings demonstrate that FcRn binding impairment of full-length IgG antibodies moderately lowers tumor accumulation of radioactivity, and shifts background activity from blood pool to liver and spleen. Furthermore, reduction of FcRn binding did not eliminate systemic click reaction, but yielded greater improvements in tumor-to-blood ratio when imaging with directly labeled antibodies than with pretargeting.


Assuntos
Anticorpos Monoclonais/química , Anticorpos Monoclonais/farmacologia , Neoplasias da Mama/tratamento farmacológico , Antígenos de Histocompatibilidade Classe I/metabolismo , Compostos Radiofarmacêuticos/metabolismo , Receptores Fc/metabolismo , Animais , Neoplasias da Mama/diagnóstico por imagem , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Química Click , Feminino , Processamento de Imagem Assistida por Computador , Camundongos , Camundongos SCID , Receptor ErbB-2/metabolismo , Tomografia Computadorizada com Tomografia Computadorizada de Emissão de Fóton Único
10.
Oncotarget ; 10(58): 6234-6244, 2019 Oct 22.
Artigo em Inglês | MEDLINE | ID: mdl-31692898

RESUMO

TENB2, a transmembrane proteoglycan protein, is a promising target for antibody drug conjugate (ADC) therapy due to overexpression in human prostate tumors and rapid internalization. We previously characterized how predosing with parental anti-TENB2 monoclonal antibody (mAb) at 1 mg/kg in a patient-derived LuCap77 explant model with high (3+) TENB2 expression could (i) block target-mediated intestinal uptake of tracer (& 0.1 mg/kg) levels of radiolabeled anti-TENB2-monomethyl auristatin E ADC while preserving tumor uptake, and (ii) maintain efficacy relative to ADC alone. Here, we systematically revisit this strategy to evaluate the effects of predosing on tumor uptake and efficacy in LuCap96.1, a low TENB2-expressing (1+) patient-derived model that is more responsive to ADC therapy than LuCap77. Importantly, rather than using tracer (& 0.1 mg/kg) levels, radiolabeled ADC tumor uptake was assessed at 1 mg/kg - one of the doses evaluated in the tumor growth inhibition study - in an effort to bridge tissue distribution (PK) with efficacy (PD). Predosing with mAb up to 1 mg/kg had no effect on efficacy. These findings warrant further investigations to determine whether predosing prior to ADC therapy might improve therapeutic index by preventing ADC disposition and possible toxicological liabilities in antigen-expressing healthy tissues.

11.
MAbs ; 10(8): 1269-1280, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30199303

RESUMO

Antibody pretargeting is a promising strategy for improving molecular imaging, wherein the separation in time of antibody targeting and radiolabeling can lead to rapid attainment of high contrast, potentially increased sensitivity, and reduced patient radiation exposure. The inverse electron demand Diels-Alder 'click' reaction between trans-cyclooctene (TCO) conjugated antibodies and radiolabeled tetrazines presents an ideal platform for pretargeted imaging due to rapid reaction kinetics, bioorthogonality, and potential for optimization of both slow and fast clearing components. Herein, we evaluated a series of anti-human epidermal growth factor receptor 2 (HER2) pretargeting antibodies containing distinct molar ratios of site-specifically incorporated TCO. The effect of stoichiometry on tissue distribution was assessed for pretargeting TCO-modified antibodies (monitored by 125I) and subsequent accumulation of an 111In-labeled tetrazine in a therapeutically relevant HER2+tumor-bearing mouse model. Single photon emission computed tomography (SPECT) imaging was also employed to assess tumor imaging at various TCO-to-monoclonal antibody (mAb) ratios. Increasing TCO-to-mAb molar ratios correlated with increased in vivo click reaction efficiency evident by increased tumor distribution and systemic exposure of 111In-labeled tetrazines. The pharmacokinetics of TCO-modified antibodies did not vary with stoichiometry. Pretargeted SPECT imaging of HER2-expressing tumors using 111In-labeled tetrazine demonstrated robust click reaction with circulating antibody at ~2 hours and good tumor delineation for both the 2 and 6 TCO-to-mAb ratio variants at 24 hours, consistent with a limited cell-surface pool of pretargeted antibody and benefit from further distribution and internalization. To our knowledge, this represents the first reported systematic analysis of how pretargeted imaging is affected solely by variation in click reaction stoichiometry through site-specific conjugation chemistry.


Assuntos
Anticorpos Monoclonais/química , Química Click/métodos , Imunoconjugados/química , Tomografia Computadorizada de Emissão de Fóton Único/métodos , Animais , Linhagem Celular Tumoral , Compostos Heterocíclicos com 1 Anel/química , Humanos , Imunoconjugados/farmacocinética , Imunoconjugados/farmacologia , Marcação por Isótopo/métodos , Camundongos , Neoplasias/diagnóstico por imagem , Neoplasias/metabolismo , Neoplasias/terapia , Radioimunoterapia/métodos , Receptor ErbB-2/imunologia , Receptor ErbB-2/metabolismo , Distribuição Tecidual , Ensaios Antitumorais Modelo de Xenoenxerto
12.
Sci Rep ; 8(1): 8239, 2018 05 29.
Artigo em Inglês | MEDLINE | ID: mdl-29844389

RESUMO

Neuregulin 1 (NRG1) is required for development of the central and peripheral nervous system and regulates neurotransmission in the adult. NRG1 and the gene encoding its receptor, ERBB4, are risk genes for schizophrenia, although how alterations in these genes disrupt their function has not been fully established. Studies of knockout and transgenic mice have yielded conflicting results, with both gain and loss of function resulting in similar behavioral and electrophysiological phenotypes. Here, we used high affinity antibodies to NRG1 and ErbB4 to perturb the function of the endogenous proteins in adult mice. Treatment with NRG1 antibodies that block receptor binding caused behavioral alterations associated with schizophrenia, including, hyper-locomotion and impaired pre-pulse inhibition of startle (PPI). Electrophysiological analysis of brain slices from anti-NRG1 treated mice revealed reduced synaptic transmission and enhanced paired-pulse facilitation. In contrast, mice treated with more potent ErbB4 function blocking antibodies did not display behavioral alterations, suggesting a receptor independent mechanism of the anti-NRG1-induced phenotypes. We demonstrate that anti-NRG1 causes accumulation of the full-length transmembrane protein and increases phospho-cofilin levels, which has previously been linked to impaired synaptic transmission, indicating enhancement of non-canonical NRG1 signaling could mediate the CNS effects.


Assuntos
Sistema Nervoso Central/fisiologia , Eletrofisiologia/métodos , Neuregulina-1/metabolismo , Esquizofrenia/metabolismo , Fatores de Despolimerização de Actina/metabolismo , Animais , Anticorpos Bloqueadores/administração & dosagem , Modelos Animais de Doenças , Predisposição Genética para Doença , Células HEK293 , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Neuregulina-1/genética , Neuregulina-1/imunologia , Estabilidade Proteica , Receptor ErbB-4/genética , Receptor ErbB-4/imunologia , Receptor ErbB-4/metabolismo , Risco , Esquizofrenia/genética , Transdução de Sinais , Transmissão Sináptica
13.
Mol Cancer Ther ; 17(4): 776-785, 2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-29339550

RESUMO

Anti-HER2/CD3, a T-cell-dependent bispecific antibody (TDB) construct, induces T-cell-mediated cell death in cancer cells expressing HER2 by cross-linking tumor HER2 with CD3 on cytotoxic T cells, thereby creating a functional cytolytic synapse. TDB design is a very challenging process that requires consideration of multiple parameters. Although therapeutic antibody design strategy is commonly driven by striving for the highest attainable antigen-binding affinity, little is known about how the affinity of each TDB arm can affect the targeting ability of the other arm and the consequent distribution and efficacy. To our knowledge, no distribution studies have been published using preclinical models wherein the T-cell-targeting arm of the TDB is actively bound to T cells. We used a combined approach involving radiochemistry, invasive biodistribution, and noninvasive single-photon emission tomographic (SPECT) imaging to measure TDB distribution and catabolism in transgenic mice with human CD3ε expression on T cells. Using CD3 affinity variants, we assessed the impact of CD3 affinity on short-term pharmacokinetics, tissue distribution, and cellular uptake. Our experimental approach determined the relative effects of (i) CD3 targeting to normal tissues, (ii) HER2 targeting to HER2-expressing tumors, and (iii) relative HER2/CD3 affinity, all as critical drivers for TDB distribution. We observed a strong correlation between CD3 affinity and distribution to T-cell-rich tissues, with higher CD3 affinity reducing systemic exposure and shifting TDB distribution away from tumor to T-cell-containing tissues. These observations have important implications for clinical translation of bispecific antibodies for cancer immunotherapy. Mol Cancer Ther; 17(4); 776-85. ©2018 AACR.


Assuntos
Anticorpos Biespecíficos/farmacologia , Anticorpos Biespecíficos/farmacocinética , Complexo CD3/imunologia , Neoplasias do Colo/tratamento farmacológico , Neoplasias do Colo/metabolismo , Receptor ErbB-2/imunologia , Linfócitos T Citotóxicos/imunologia , Animais , Afinidade de Anticorpos , Neoplasias do Colo/patologia , Modelos Animais de Doenças , Feminino , Humanos , Imunoterapia , Camundongos , Camundongos Nus , Camundongos Transgênicos , Linfócitos T Citotóxicos/efeitos dos fármacos , Linfócitos T Citotóxicos/patologia , Distribuição Tecidual , Células Tumorais Cultivadas
14.
MAbs ; 8(3): 593-603, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-26918260

RESUMO

MPDL3280A is a human monoclonal antibody that targets programmed cell death-1 ligand 1 (PD-L1), and exerts anti-tumor activity mainly by blocking PD-L1 interaction with programmed cell death-1 (PD-1) and B7.1. It is being investigated as a potential therapy for locally advanced or metastatic malignancies. The purpose of the study reported here was to characterize the pharmacokinetics, pharmacodynamics, tissue distribution and tumor penetration of MPDL3280A and/or a chimeric anti-PD-L1 antibody PRO304397 to help further clinical development. The pharmacokinetics of MPDL3280A in monkeys at 0.5, 5 and 20 mg · kg(-1) and the pharmacokinetics / pharmacodynamics of PRO304397 in mice at 1, 3 10 mg · kg(-1) were determined after a single intravenous dose. Tissue distribution and tumor penetration for radiolabeled PRO304397 in tumor-bearing mouse models were determined. The pharmacokinetics of MPDL3280A and PRO304397 were nonlinear in monkeys and mice, respectively. Complete saturation of PD-L1 in blood in mice was achieved at serum concentrations of PRO304397 above ∼ 0.5 µg · mL(-1). Tissue distribution and tumor penetration studies of PRO304397 in tumor-bearing mice indicated that the minimum tumor interstitial to plasma radioactivity ratio was ∼ 0.3; saturation of target-mediated uptake in non-tumor tissues and desirable exposure in tumors were achieved at higher serum concentrations, and the distribution into tumors was dose-and time-dependent. The biodistribution data indicated that the efficacious dose is mostly likely higher than that estimated based on simple pharmacokinetics/pharmacodynamics in blood. These data also allowed for estimation of the target clinical dose for further development of MPDL3280A.


Assuntos
Anticorpos Monoclonais , Anticorpos Antineoplásicos , Antígeno B7-H1/antagonistas & inibidores , Pontos de Checagem do Ciclo Celular , Proteínas de Neoplasias/antagonistas & inibidores , Neoplasias Experimentais , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/farmacocinética , Anticorpos Monoclonais/farmacologia , Anticorpos Antineoplásicos/imunologia , Anticorpos Antineoplásicos/farmacologia , Antígeno B7-H1/imunologia , Células CHO , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Pontos de Checagem do Ciclo Celular/imunologia , Cricetinae , Cricetulus , Avaliação Pré-Clínica de Medicamentos , Feminino , Humanos , Macaca fascicularis , Camundongos , Camundongos Endogâmicos BALB C , Metástase Neoplásica , Proteínas de Neoplasias/imunologia , Neoplasias Experimentais/tratamento farmacológico , Neoplasias Experimentais/imunologia , Neoplasias Experimentais/patologia
15.
MAbs ; 6(5): 1255-64, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25517310

RESUMO

Pharmacokinetic (PK) testing of a humanized (κI, VH3 framework) and affinity matured anti-hepatitis C virus E2-glycoprotein (HCV-E2) antibody (hu5B3.κ1VH3.v3) in rats revealed unexpected fast clearance (34.9 mL/day/kg). This antibody binds to the rat recycling receptor FcRn as expected for a human IgG1 antibody and does not display non-specific binding to baculovirus particles in an assay that is correlated with fast clearance in cynomolgus monkey. The antigen is not expressed in rat so target-dependent clearance does not contribute to PK. Removal of the affinity maturation changes (hu5B3.κ1VH3.v1) did not restore normal clearance. The antibody was re-humanized on a κ4, VH1 framework and the non-affinity matured version (hu5B3.κ4VH1.v1) was shown to have normal clearance (8.5 mL/day/kg). Since the change in framework results in a lower pI, primarily due to more negative charge on the κ4 template, the effect of additional charge variation on antibody PK was tested by incorporating substitutions obtained through phage display affinity maturation of hu5B3.κ1VH3.v1. A variant having a pI of 8.61 gave very fast clearance (140 mL/day/kg) whereas a molecule with pI of 6.10 gave slow clearance (5.8 mL/kg/day). Both antibodies exhibited comparable binding to rat FcRn, but biodistribution experiments showed that the high pI variant was catabolized in liver and spleen. These results suggest antibody charge can have an effect on PK through alterations in antibody catabolism independent of FcRn-mediated recycling. Furthermore, introduction of affinity maturation changes into the lower pI framework yielded a candidate with PK and virus neutralization properties suitable for clinical development.


Assuntos
Anticorpos Monoclonais Humanizados/imunologia , Anticorpos Monoclonais Humanizados/farmacocinética , Imunoglobulina G/imunologia , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais Humanizados/genética , Área Sob a Curva , Sítios de Ligação/genética , Sítios de Ligação/imunologia , Ensaio de Imunoadsorção Enzimática , Antígenos de Histocompatibilidade Classe I/imunologia , Antígenos de Histocompatibilidade Classe I/metabolismo , Humanos , Imunoglobulina G/química , Imunoglobulina G/metabolismo , Macaca fascicularis , Taxa de Depuração Metabólica , Modelos Moleculares , Dados de Sequência Molecular , Ligação Proteica/imunologia , Estrutura Terciária de Proteína , Ratos Sprague-Dawley , Receptores Fc/imunologia , Receptores Fc/metabolismo , Homologia de Sequência de Aminoácidos , Distribuição Tecidual
16.
MAbs ; 6(6): 1631-7, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25484068

RESUMO

Delta-like-4 ligand (DLL4) plays an important role in vascular development and is widely expressed on the vasculature of normal and tumor tissues. Anti-DLL4 is a humanized IgG1 monoclonal antibody against DLL4. The purpose of these studies was to characterize the pharmacokinetics (PK), tissue distribution, and anti-tumor efficacy of anti-DLL4 in mice over a range of doses. PK and tissue distribution of anti-DLL4 were determined in athymic nude mice after administration of single intravenous (IV) doses. In the tissue distribution study, radiolabeled anti-DLL4 (mixture of (125)Iodide and (111)Indium) was administered in the presence of increasing amounts of unlabeled anti-DLL4. Dose ranging anti-DLL4 anti-tumor efficacy was evaluated in athymic nude mice bearing MV522 human lung tumor xenografts. Anti-DLL4 had nonlinear PK in mice with rapid serum clearance at low doses and slower clearance at higher doses suggesting the involvement of target mediated clearance. Consistent with the PK data, anti-DLL4 was shown to specifically distribute to several normal tissues known to express DLL4 including the lung and liver. Maximal efficacy in the xenograft model was seen at doses ≥ 10 mg/kg when tissue sinks were presumably saturated, consistent with the PK and tissue distribution profiles. These findings highlight the importance of mechanistic understanding of antibody disposition to enable dosing strategies for maximizing efficacy.


Assuntos
Anticorpos Monoclonais Humanizados/farmacocinética , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/metabolismo , Proteínas de Membrana/antagonistas & inibidores , Animais , Anticorpos Monoclonais Humanizados/sangue , Anticorpos Monoclonais Humanizados/imunologia , Área Sob a Curva , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Feminino , Humanos , Imunoglobulina G/sangue , Imunoglobulina G/imunologia , Imunoglobulina G/farmacologia , Radioisótopos de Índio/farmacocinética , Peptídeos e Proteínas de Sinalização Intracelular/imunologia , Radioisótopos do Iodo/farmacocinética , Neoplasias Pulmonares/imunologia , Proteínas de Membrana/imunologia , Taxa de Depuração Metabólica , Camundongos Nus , Distribuição Tecidual , Resultado do Tratamento , Ensaios Antitumorais Modelo de Xenoenxerto
17.
MAbs ; 6(3): 689-96, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24572100

RESUMO

The neonatal Fc receptor (FcRn) plays an important and well-known role in antibody recycling in endothelial and hematopoietic cells and thus it influences the systemic pharmacokinetics (PK) of immunoglobulin G (IgG). However, considerably less is known about FcRn's role in the metabolism of IgG within individual tissues after intravenous administration. To elucidate the organ distribution and gain insight into the metabolism of humanized IgG1 antibodies with different binding affinities FcRn, comparative biodistribution studies in normal CD-1 mice were conducted. Here, we generated variants of herpes simplex virus glycoprotein D-specific antibody (humanized anti-gD) with increased and decreased FcRn binding affinity by genetic engineering without affecting antigen specificity. These antibodies were expressed in Chinese hamster ovary cell lines, purified and paired radiolabeled with iodine-125 and indium-111. Equal amounts of I-125-labeled and In-111-labeled antibodies were mixed and intravenously administered into mice at 5 mg/kg. This approach allowed us to measure both the real-time IgG uptake (I-125) and cumulative uptake of IgG and catabolites (In-111) in individual tissues up to 1 week post-injection. The PK and distribution of the wild-type IgG and the variant with enhanced binding for FcRn were largely similar to each other, but vastly different for the rapidly cleared low-FcRn-binding variant. Uptake in individual tissues varied across time, FcRn binding affinity, and radiolabeling method. The liver and spleen emerged as the most concentrated sites of IgG catabolism in the absence of FcRn protection. These data provide an increased understanding of FcRn's role in antibody PK and catabolism at the tissue level.


Assuntos
Anticorpos Monoclonais Humanizados/farmacocinética , Receptores Fc/metabolismo , Animais , Animais Recém-Nascidos , Anticorpos Monoclonais Humanizados/administração & dosagem , Anticorpos Monoclonais Humanizados/genética , Células CHO , Cricetinae , Cricetulus , Feminino , Variação Genética , Humanos , Imunoglobulina G/administração & dosagem , Imunoglobulina G/genética , Imunoglobulina G/metabolismo , Radioisótopos de Índio/administração & dosagem , Radioisótopos de Índio/farmacocinética , Radioisótopos do Iodo/administração & dosagem , Radioisótopos do Iodo/farmacocinética , Camundongos , Proteólise , Receptores Fc/genética , Distribuição Tecidual
18.
J Med Chem ; 56(23): 9418-26, 2013 Dec 12.
Artigo em Inglês | MEDLINE | ID: mdl-24131491

RESUMO

A known limitation of iodine radionuclides for labeling and biological tracking of receptor targeted proteins is the tendency of iodotyrosine to rapidly diffuse from cells following endocytosis and lysosomal degradation. In contrast, radiometal-chelate complexes such as indium-111-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (In-111-DOTA) accumulate within target cells due to the residualizing properties of the polar, charged metal-chelate-amino acid adduct. Iodine radionuclides boast a diversity of nuclear properties and chemical means for incorporation, prompting efforts to covalently link radioiodine with residualizing molecules. Herein, we describe the Ugi-assisted synthesis of [I-125]HIP-DOTA, a 4-hydroxy-3-iodophenyl (HIP) derivative of DOTA, and demonstration of its residualizing properties in a murine xenograft model. Overall, this study displays the power of multicomponent synthesis to yield a versatile radioactive probe for antibodies across multiple therapeutic areas with potential applications in both preclinical biodistribution studies and clinical radioimmunotherapies.


Assuntos
Anticorpos Monoclonais Murinos/metabolismo , Dipeptídeos/síntese química , Compostos Heterocíclicos com 1 Anel/síntese química , Imunoconjugados/química , Succinimidas/síntese química , Animais , Anticorpos Monoclonais Murinos/química , Anticorpos Monoclonais Murinos/uso terapêutico , Complexos de Coordenação/metabolismo , Dipeptídeos/metabolismo , Compostos Heterocíclicos com 1 Anel/metabolismo , Imunoconjugados/metabolismo , Radioisótopos de Índio , Camundongos , Radioimunoterapia , Succinimidas/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
19.
Br J Pharmacol ; 168(2): 445-57, 2013 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-22889168

RESUMO

BACKGROUND AND PURPOSE: The success of antibody-drug conjugates (ADCs) depends on the therapeutic window rendered by the differential expression between normal and pathological tissues. The ability to identify and visualize target expression in normal tissues could reveal causes for target-mediated clearance observed in pharmacokinetic characterization. TENB2 is a prostate cancer target associated with the progression of poorly differentiated and androgen-independent tumour types, and ADCs specific for TENB2 are candidate therapeutics. The objective of this study was to locate antigen expression of TENB2 in normal tissues, thereby elucidating the underlying causes of target-mediated clearance. EXPERIMENTAL APPROACH: A series of pharmacokinetics, tissue distribution and mass balance studies were conducted in mice using a radiolabelled anti-TENB2 ADC. These data were complemented by non-invasive single photon emission computed tomography - X-ray computed tomography imaging and immunohistochemistry. KEY RESULTS: The intestines were identified as a saturable and specific antigen sink that contributes, at least in part, to the rapid target-mediated clearance of the anti-TENB2 antibody and its drug conjugate in rodents. As a proof of concept, we also demonstrated the selective disposition of the ADC in a tumoural environment in vivo using the LuCaP 77 transplant mouse model. High tumour uptake was observed despite the presence of the antigen sink, and antigen specificity was confirmed by antigen blockade. CONCLUSIONS AND IMPLICATIONS: Our findings provide the anatomical location and biological interpretation of target-mediated clearance of anti-TENB2 antibodies and corresponding drug conjugates. Further investigations may be beneficial in addressing the relative contributions to ADC disposition from antigen expression in both normal and pathological tissues.


Assuntos
Antígenos/imunologia , Compostos Heterocíclicos com 1 Anel/farmacocinética , Imunoconjugados/farmacocinética , Proteínas de Membrana/imunologia , Proteínas de Neoplasias/imunologia , Oligopeptídeos/farmacocinética , Animais , Linhagem Celular Tumoral , Citotoxinas/química , Compostos Heterocíclicos com 1 Anel/química , Imunoconjugados/química , Masculino , Camundongos , Camundongos SCID , Neoplasias/metabolismo , Oligopeptídeos/química , Preparações Farmacêuticas , Distribuição Tecidual
20.
J Nucl Med ; 53(9): 1454-61, 2012 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-22872740

RESUMO

UNLABELLED: TENB2, also known as tomoregulin or transmembrane protein with epidermal growth factor-like and 2 follistatin-like domains, is a transmembrane proteoglycan overexpressed in human prostate tumors. This protein is a promising target for antimitotic monomethyl auristatin E (MMAE)-based antibody-drug conjugate (ADC) therapy. Nonlinear pharmacokinetics in normal mice suggested that antigen expression in normal tissues may contribute to targeted mediated disposition. We evaluated a predosing strategy with unconjugated antibody to block ADC uptake in target-expressing tissues in a mouse model while striving to preserve tumor uptake and efficacy. METHODS: Unconjugated, unlabeled antibody was preadministered to mice bearing the TENB2-expressing human prostate explant model, LuCaP 77, followed by a single administration of (111)In-labeled anti-TENB2-MMAE for biodistribution and SPECT/CT studies. A tumor-growth-inhibition study was conducted to determine the pharmacodynamic consequences of predosing. RESULTS: Preadministration of anti-TENB2 at 1 mg/kg significantly increased blood exposure of the radiolabeled ADC and reduced intestinal, hepatic, and splenic uptake while not affecting tumor accretion. Similar tumor-to-heart ratios were measured by SPECT/CT at 24 h with and without the predose. Consistent with this, the preadministration of 0.75 mg/kg did not interfere with efficacy in a tumor-growth study dosed at 0.75 mg or 2.5 mg of ADC per kilogram. CONCLUSION: Overall, the potential to mask peripheral, nontumor antigen uptake while preserving tumor uptake and efficacy could ameliorate toxicity and may significantly affect future dosing strategies for ADCs.


Assuntos
Anticorpos/farmacologia , Imunoconjugados/imunologia , Imunoconjugados/metabolismo , Radioisótopos de Índio/uso terapêutico , Proteínas de Membrana/imunologia , Proteínas de Neoplasias/imunologia , Neoplasias da Próstata/metabolismo , Animais , Anticorpos/imunologia , Transporte Biológico/efeitos dos fármacos , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Humanos , Imunoconjugados/uso terapêutico , Marcação por Isótopo , Masculino , Camundongos , Imagem Multimodal , Tomografia por Emissão de Pósitrons , Neoplasias da Próstata/diagnóstico por imagem , Neoplasias da Próstata/patologia , Tomografia Computadorizada por Raios X
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